Based on the validation dataset (Versteeg), we confirmed that CTSL served as a prognostic marker for poor clinical outcome in NB patients. We further found that the expression level of CTSL was higher in SK-N-BE (2) cells than in IMR-32 cells. Knocking down CTSL reversed the chemoresistance in SK-N-BE (2) cells. Furthermore, combination of CTSL inhibition and chemotherapy potently blocked tumor growth in vivo. Mechanistically, CTSL promoted chemoresistance in NB cells by up-regulating multi-drug resistance protein ABCB1 and ABCG2, inhibiting the autophagy level and cell apoptpsis. Furthermore, we observed six datasets and found that Serglycin (SRGN) expression was positively associated with CTSL expresssion. CTSL could mediate chemoresistance by up-regulating SRGN expression in NB cells and SRGN expression was positively correlated with poor prognosis of NB patients. Taken together, our findings indicate that the CTSL promotes chemoresistance to cisplatin and doxorubicin by up-regulating the expression of multi-drug resistance proteins and inhibiting the autophagy level and cell apoptosis in NB cells. Thus, CTSL may be a therapeutic target for overcoming chemoresistant to cisplatin and doxorubicin in NB patients.Cathepsin L (CTSL), a lysosomal acid cysteine protease, is found to play a critical role in chemosencitivity and tumor progression. However, the potential roles and molecular mechanisms of CTSL in chemoresistance in neuroblastoma (NB) are still unclear. In this study, the correlation between clinical characteristics, survival and CTSL expression were assessed in Versteeg dataset. The chemoresistant to cisplatin or doxorubicin was detected using CCK-8 assay. Western blot was employed to detect the expression of CTSL, multi-drug resistance proteins, autophagy-related proteins and apoptosis-related proteins in NB cells while knocking down CTSL. Lysosome staining was analyzed to access the expression levels of lysosomes in NB cells. The expression of apoptosis markers was analyzed with immunofluorescence. Various datasets were analyzed to find the potential protein related to CTSL. In addition, a subcutaneous tumor xenografts model in M-NSG mice was used to assess tumor response to CTSL inhibition  Neuroblastoma (NB) is the most common extracranial solid tumor in children. NB accounts for 10% of all childhood cancers and also 15% of pediatric cancer deaths . Main avVarious molecular mechanisms have been implicated in chemoresistance. The ATP-binding cassette transporter (ABC) family of transmembrane proteins is associated with chemoresistance by promoting drug efflux. Among these, ABC transporter B1 (ABCB1/MDR1/P-glycoprotein), ABC transporter C1 (ABCC1/MRP1) and breast cancer resistance protein (ABCG2/BCRP) are closely related to poor platinum sensitivity . ResistaThe ability of cancer cells to maintain an internal stasis is a critical characteristic of a neoplasm . The impIn the present study, we demonstrated that CTSL was a regulator of poor DDP and ADM sensitivity in NB cells, and the regulation of chemoresistance by CTSL was mediated through its effects on ABC proteins, autophagy and cell apoptosis. These findings indicate that CTSL may represent a novel therapeutic target to overcome poor DDP and ADM sensitivity in NB patients.http://r2.amc.nl) using the following publicly available dataset: Versteeg (GEO: GSE16476)  , which ip < 0.001 and at the top 20 of the datasets, the eligible common genes in six databases could be screened out. Then the Spearman correlation analysis between the selected gene and CTSL was performed.Six datasets  were observed at the same time and different colors represented different datasets in the box plot after standardization, According to −0.6 < R < 0.6 and The human NB cell lines, SK-N-BE (2) and IMR-32 were obtained from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. SK-N-BE (2) and IMR-32 cells were cultured in a 1:1 mixture of MEM and DMEM/F-12 with 10% fetal bovine serum, 1% Penicillin-Streptomycin. These cells were placed in an incubator with 5% CO₂ at 37°C and passaged every 72 h.4/ml, were inoculated into 96-well culture plates with 100 μl/well, cultured overnight in an incubator with 5% CO₂ at 37°C and treated the next day. After pretreatment with different concentrations of DDP  or ADM  for 24 h, 10 μl CCK-8 solution was added to each well and incubated for 4 h at 37°C. The optical density was measured at 450 nm. All assays were performed in triplicate.Cell Counting Kit-8 (CCK-8) assay was used to measure the viability and proliferation of cells. SK-N-BE (2) and IMR-32 cells in logarithmic growth phase, with the appropriate concentration of 5 × 10® 3000 Reagent (Invitrogen) and then transfected into SK-N-BE (2) or IMR-32 cells. After 6 h, the supernatant was replaced with fresh medium containing 10% FBS and cultured for another 24 h. Three siRNA sequences were used for transfection . For transfection, siRNA was mixed with Lipofectaminesfection .A lentivirus carrying CTSL gene was constructed by GeneChem . IMR-32 cells were seeded in 6-well plate and then infected with the lentivirus according to protocols as recommended by the manufacturer. After 16 h, the medium was replaced with complete medium. In order to obtain a stable CTSL overexpressing cell line, the lentivirus infected cells were selected by incubation with complete medium of 2 μg/ml puromycin. The expression of CTSL in IMR-32 cell lines stably infected with a lentivirus was examined by Western blot.Detailed procedure was as described in a previous study . PrimaryDetailed procedure for these steps has been previously reported . The priCells were cultured on glass coverslips and then fixed with 4% paraformaldehyde. After a PBS wash, the cells were permeabilized employing 0.1% Triton X-100, incubated in a blocking solution , then further incubated overnight at 4°C with the primary antibody to Bcl-2, SRGN (Santa Cruz Biotechnology) and CTSL (Abcam). The fluorescent conjugated secondary antibodies were Alexa Fluor 488 and Alexa Fluor 594 , and 4′,6-diamidino-2-phenylindole (DAPI)  was employed as a nuclear counterstain for 10 min. The coverslips were finally mounted onto slides with fluorescent mounting medium and instantly observed by confocal microscopy.After removing the cell culture solution, the lysosome green fluorescent probe staining solution  was added, and the lysosome green fluorescent probe solution and the cell culture solution were mixed at a ratio of 1/20,000, and then incubated with cells in an incubator with 5% CO₂ at 37°C for 60 min. The staining solution was discarded and new cell culture solution was added. Finally the cells were observed under the laser confocal microscope.Apoptotic cell death was assessed by flow cytometry using the AnnexinV-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit (KeyGEN). Cells were harvested and resuspended in binding buffer, then labeled with Annexin V-FITC/PI reagent for 15 min in the dark at room temperature. For each analysis, a minimum of 20,000 cells per sample were analyzed using a Beckman Coulter FACS machine. Results were analyzed and calculated by FlowJo V.10 software; the percentage of apoptosis was obtained from the sum of (Annexin V-FITC+/PI−) and (Annexin V-FITC+/PI+) cells.scidIl2rgem1/Smoc) mice  were used in the study. Subcutaneous tumor transplantation was conducted using the SK-N-BE (2) cells. Cells (n = 1 × 107) were resuspended in 100 μl PBS and implanted into the right flank of nude mice under sterile conditions. After the formation of palpable tumors (tumor volume reached 100 mm3), mice were randomized into four groups (5 mice per group): control group , Z-FY-CHO  group , ADM group , Z-FY-CHO  plus ADM  group. Mice were injected with vehicle or with drugs three times weekly. The size of the tumor and the body weight of each mouse were measured as described previously  test or Fisher exact test was used to compare qualitative variables. Differences were considered statistically significant at p values of <0.05. All analyses were performed employing GraphPad Prism 5.0.All measurement data were expressed as the mean ± S.D. at least three independent experiments were conducted. Differences in measured variables between the experimental and control groups were assessed by Student’s p = 0.0071; p = 0.0161; To figure out the relationship between the expression of CTSL and prognosis of NB patients, we analyzed the public dataset Versteeg to examine the correlation between the mRNA expression of CTSL and the survival rates in NB. In the Versteeg dataset, patients with high CTSL mRNA levels (cut off: 615.00) showed significantly poor overall survival (OS)  cells . CCK-8 ain vivo. The subcutaneous tumor xenograft model was established using SK-N-BE (2) cells, and then treated with Z-FY-CHO or ADM. As shown in 3 in the control group, 1850.10 ± 255.30 mm3 in Z-FY-CHO group, 1124.80 ± 343.89 mm3 in ADM group, and 344.60 ± 156.03 mm3 in Z-FY-CHO plus ADM group. The average volumes of tumors were remarkably decreased in Z-FY-CHO plus ADM group compared to ADM group (p < 0.05), which was consistent with the results of experiments in vitro. The weights of mice were also recorded. As was shown, there was no significant difference among those groups in tumor weight  cells, we chose si-CTSL (450) with the best interference effect to interfere the CTSL expression of SK-N-BE (2) cells . CCK-8 ar weight . The resTo explore the mechanisms of chemoresistance in NB cells with high CTSL expression, Western blot was used to detect the expression level of ABCB1 and ABCG2 in NC group and si-CTSL group. It turned out that the expression levels of ABCB1 and ABCG2 were lower in si-CTSL group than that of NC group . The resp < 0.001). Then, the expression levels of Bcl-2 protein and Bax protein were detected by western blot to determine whether knockdown of CTSL could induce cell apoptosis. We found that the expression level of anti-apoptotic protein Bcl-2 in si-CTSL group under the action of ADM and DDP with the same concentrations was lower than that of NC group, and the expression level of apoptosis-related protein Bax in si-CTSL group under the action of ADM and DDP with the same concentrations was higher than that of NC group  cells with different treatments. Apoptosis levels did not significantly increase in si-CTSL (450) group compared with NC group ; by contNC group , which iNC group . These rp < 0.001; P: 0.01577; 95% CI: 0.10–0.27; p = 0.0014; p = 0.0430; p < 0.001; To further explore the downstream targets of CTSL in NB cells, six datasets were observed at the same time and showed that only Serglycin (SRGN) expression was associated with the expression levels of CTSL  cells showed higher expression of CTSL and more resistant to ADM or DDP. These results indicated that CTSL might mediate the decrease of ADM and DDP sensitivity in SK-N-BE (2) cells. In order to verify the role of CTSL in mediating chemoresistance, we used CTSL targeting si-RNAs to interfere CTSL, and the chemoresistance of SK-N-BE (2) cells to ADM decreased by about 1.7 times and that of SK-N-BE (2) cells to DDP decreased by about 2.6 times. Similarly, cotreatment with CTSL inhibition and ADM in M-NSG mice of subcutaneous tumors exhibited less tumor growth. To further verify the role of CTSL in regulating the decrease of the sensitivity to ADM and DDP, we detected the expression levels of multidrug resistance proteins ABCB1 and ABCG2 by western blot and found that ABCB1 and ABCG2 expression were decreased in si-CTSL group, which indicated that the sensitivity of SK-N-BE (2) cells to ADM and DDP were significantly increased after si-RNA interfering CTSL. According to the above results, it was demonstrated that CTSL was involved in regulating the decrease of ADM and DDP sensitivity in SK-N-BE (2) cells.To explore the mechanisms of CTSL in the chemoresistance of SK-N-BE (2) cells, the autophagy of cells was observed from the cell morphology, and the green fluorescent spots of SK-N-BE (2) cells in si-CTSL group were brighter and more, indicating that the autophagy level was higher. With autophagy protein LC3-II as an index, we observed that the autophagy level of SK-N-BE (2) cells in si-CTSL group increased significantly under the treatment of ADM and DDP. After si-RNA interfering CTSL, the expression level of pro-apoptotic protein Bax increased and the expression level of anti-apoptotic protein Bcl-2 decreased under the treatment of ADM and DDP. The above results indicated that CTSL could mediate the chemoresistance of NB by regulating the autophagy and apoptosis of SK-N-BE (2) cells.SRGN is a low molecular weight glycoprotein, which plays a critical role in the storage and secretion of some chemokines, cytokines and proteases, thus, it participates in lots of physiological and pathological processes . SRGN haThe study still has some limitations. Since this study has not constructed NB chemoresistant cells, we have not been able to clarify the role of CTSL in NB chemothresistant cells, which needed to be further verified by more experiments. Meanwhile, the mechanism about CTSL how to regulate SRGN needed to be clarified. As reported before, CTSL could get into the nucleus, and then processes and activates certain transcription factors to perform transcription functions . It was In all, our study explored the role and molecular mechanism of CTSL in regulating the chemoresistance of ADM and DDP in NB cells, and found that CTSL could mediate the chemoresistance of NB by up-regulating the expression of SRGN. Therefore, CTSL seems to play a key role in improving the chemotherapy sensitivity of NB, and it may become an important target to improve the chemosensitivity of NB and the effect of ADM and DDP.


Glaucosciadium cordifolium essential oil  on anxiety and learning and memory impairment induced by scopolamine (SCOP) in zebrafish. The chemical composition was analyzed by GC-MS, and the results showed that the highest content was limonene followed by α- and β-pinene, p-cymene and α-phellandrene. The dementia model was induced by SCOP (100 µM), whereas GCEO and galantamine  were delivered to the SCOP-induced model. It was found that GCEO significantly improved memory impairment and anxiety-like response induced by SCOP through the Y-maze, novel object recognition (NOR) test, and novel tank diving tests (NTT). Biochemical analyses showed that GCEO reduced SCOP-induced oxidative damage. Additionally, the cholinergic system activity was improved in the SCOP-induced model by decreasing the acetylcholinesterase (AChE) activity following the exposure to GCEO. It was clear that as a mixture, GCEO displays positive action in improving memory impairment through restoring cholinergic dysfunction and brain antioxidant status.The purpose of this study was to investigate the effect of  Alzheimer’s disease (AD) is a progressive, unremitting brain disease and a major cause of dementia . Due to Danio rerio) is a small, hardy freshwater fish that originated from India and has emerged as an excellent complementary model for neurodegenerative research due to the simplicity of the organism and robust and clearly visible behavior forms. Zebrafish is also a great translational model between in vitro and in vivo mammalian models for drug screening and for studying vertebrate development  × 100. The cubes had sides of 2.5 cm in length, were made of non-toxic plastic and were colored in yellow or blue, as the zebrafish show no innate preference for these colors [The recognition memory of the zebrafish, i.e., the ability of these to recognize novel objects in the environment, was assessed in the novel object recognition (NOR) test, as previously described by Gaspary et al.  and Stefe colors . The expe colors . Therefow/v) in ice-cold 0.1 M potassium phosphate buffer with 1.15% KCl (pH 7.4), for 1 min at 1000 rpm using the Mikro-Dismembrator U mill  equipped with 3 mm diameter magnetic balls . Then, the crude homogenates were centrifuged for 15 min at 14,000 rpm and 4 °C, and the supernatants were further used to measure the total content of soluble proteins (bicinchoninic acid assay) [Once the behavioral tasks were finished, the zebrafish were euthanized by rapid cooling. It is known that this method avoids the biochemical and physiological alterations that could prevent post-mortem analysis . The anid assay) , the actd assay) , CAT (ECd assay) , GPX (ECd assay) , AChE (Ed assay)  and the d assay)  and carbd assay) .p < 0.05. Pearson correlation coefficient (r) was applied to correlate the behavioral and biochemical results.The data were expressed as means ± standard error of mean (S.E.M.) and were statistically analyzed with GraphPad Prism v9.4 software  using one-way analysis of variance (one-way ANOVA) and Tukey’s post hoc multiple comparison tests. Statistically significant differences were considered when The current study was conducted to investigate the promnesic, anxiolytic and antioxidant potential of GCEO in a zebrafish model of cognitive impairment induced by SCOP. Our data demonstrated that GCEO administration mitigated the memory deficits and the anxiety-like behavior measured in the specific in vivo tasks. In addition, the observed effects could be attributed to the main identified compounds such as limonene, α- and β-pinene, p-cymene and α-phellandrene. Moreover, the chronic treatment with GCEO reduced the SCOP-induced oxidative stress and displayed a pronounced anti-AChE effect. This study suggests, for the first time, that GCEO possesses promnesic, anxiolytic and antioxidant effects that could be used effectively in amelioration of dementia-related conditions.


It is controversial whether donor-recipient sex mismatch is a risk factor associated with corneal graft failure. The purpose of this study was to investigate the effect of sex mismatch on corneal graft failure in high-risk and non-high-risk patients.  A retrospective study.  The medical charts of patients who underwent corneal transplantations by one surgeon between 2012 and 2017 were reviewed. Patients were defined as high-risk for failure if they had glaucoma, ocular surface disease, or corneal vascularization. Graft failure rates were compared using the Kaplan–Meier survival curves between sex matched and mismatched subjects and between male-to-female grafting and other patients. p=0.645, log-rank) and in male donor to female recipient transplantations and in the other transplantations . Analysis of fifty-one eyes of 51 high-risk graft recipients  showed that graft failure rates were significantly higher in the sex mismatched than sex matched recipients  and in male donor to female recipient transplantations than in the other transplantations .  One hundred and thirteen patients with a minimum follow-up of 18 months were included. In 62 non-high-risk patients, graft failure rates were similar between the sex mismatched and the sex matched recipients ( Sex matching for every patient bares logistic difficulties; however, in patients who are at high-risk for graft failure, it may be a simple way to improve outcomes and better utilize corneal grafts. The cornea is an immune-privileged site due to various factors, including the absence of blood and lymphatic vessels, blood-eye barrier, and immunomodulatory factors. Thus, any cornea deemed appropriate for transplantation can be transplanted into any recipient, regardless of the recipient's age, sex, and HLA type . HoweverMale-to-female (M⟶F) corneal grafts have been reported to have a lower graft survival rate compared to matched corneal transplants . This fiIn low-risk patients undergoing Descemet's stripping endothelial keratoplasty (DSEK) or Descemet membrane endothelial keratoplasty (DMEK), where the risk for failure is low, sex mismatching was not associated with graft survival . A recenHigh-risk keratoplasty is associated with low success rates due to a high incidence of immune-mediated graft rejection  and may A retrospective chart review of all patients of age ≥18 years who underwent primary corneal transplantation at the E. Wolfson Medical Center, Sackler Faculty of Medicine, Tel Aviv University, between 2012 and 2017 was carried out. DMEK was performed at our institute starting in 2017. The steep learning curve associated with this procedure may bias the results. Thus, DMEK cases done in 2017 were not reviewed.Primary graft failures (nonclearing grafts within 2 weeks of transplantation) were excluded as they represent complexities with donor tissues unrelated to donor-recipient matching . Deep anA single surgeon (HA) performed all surgeries. Postoperative visits were scheduled for day 1; weeks 1, 2, and 4; and months 3 and 6, and then every 6 months. Topical antibiotics t.i.d. and corticosteroid drops (dexamethasone 0.1%) t.i.d. were applied after PK and DSEK. Antibiotics were stopped after one week and corticosteroid drops were tapered down, usually to b.i.d. after one month and once daily after an additional 3 months. After that, a less potent corticosteroid (fluorometholone 0.1%) was used once daily. In high-risk patients, usually a more potent corticosteroid (dexamethasone 0.1%) is used once daily.Graft failure was defined according to the cornea preservation time study as the occurrence of a corneal graft that was initially clear and subsequently became cloudy, preventing recovery of useful vision and requiring additional corneal transplantation . FailureFailure-free graft survival was defined as the period from keratoplasty to the postoperative meeting in which the patient was diagnosed with failure. The study was approved by the Institutional Review Board of the E. Wolfson Medical Center and complied with the principles outlined in the Declaration of Helsinki.t-test and nonparametric variables with the Mann–Whitney U test. Categorical data was analyzed with Pearson's chi-square test or Fisher's exact test when values in any of the cells of a contingency table were below five. The donor-recipient relationship was divided according to sex matched status. The sex matched group included male to male (M⟶M) and female to female (F⟶F) donations, while the sex mismatched group included male to female (M⟶F) and female to male (F⟶M) donations. For survival, the Kaplan-Meier analysis with log-rank (Mantel–Cox) was applied. IBM SPSS Statistics 27  was used for statistical analysis. Unless otherwise specified, values represent the mean ± standard deviation.The clinical parameter distribution for normality was analyzed by the Shapiro–Wilk test. For two-group comparisons, parametric variables were analyzed with the Student's One hundred and thirteen eyes of 113 patients with a mean follow-up time of 3.2 ± 1.3 years were included.p=0.645). No difference was observed in graft failure among the male donor to female recipient transplantations when compared to other transplantations (p=0.496).In the 62 non-high-risk patients, the mean donor and recipient ages were 63.0 ± 9.1 and 75.1 ± 9.7 years, respectively. Male-to-female sex distribution was 47 : 15 (76 : 24%) and 18 : 44 (29 : 71%), among donors and recipients, respectively. There were 23 (37.1%) cases of sex-matched transplantations. Over a mean follow-up time of 3.2 ± 1.3 years, 6 (9.7%) cases of graft failure were documented. No difference was observed in graft failure among the sex mismatched when compared to sex matched recipients  and 25 : 26 (49 : 51%) among donors and recipients, respectively.DSEK was performed in 26 (51%) cases and penetrating keratoplasty (PK) in 25 (49%) cases. There were 33 (65%) cases of sex-matched transplantations. Over a mean follow-up time of 3.0 ± 1.4 years, 11 (21.6%) cases of graft failure were documented.There were no differences in the baseline characteristics between the sex match and mismatch groups . Baselinp=0.022; p=0.002; The graft failure rate was significantly higher among the sex-mismatched when compared to sex matched recipients p=0.022; . The grap=0.034, A comparison of baseline characteristics between non-high-risk and high-risk patients is shown in In this study, we found that in high-risk patients having comorbidities of glaucoma, ocular surface disease, or corneal vascularization, sex mismatching was associated with an increased risk for graft failure after a mean of three years of follow-up. This was emphasized especially in the male-to-female corneal transplantations. Thus, according to our study, women with high-risk characteristics for corneal graft failure, facing corneal transplantation, could benefit from female donors.Currently, corneal transplantation guidelines do not support corneal matching due to the extensive immune privilege of the cornea . The immp=0.02). Hopkinson et al. [In 2006, Böhringer et al,  were then et al.  reportedn et al.  showed tn et al. . A recenn et al. . An updan et al. . In a ren et al. .Regarding the effect of sex mismatching on corneal graft outcomes in high-risk patients, data is sparse. Hopkinson et al. have reported that in high-risk patients, sex mismatching results in a higher graft failure rate, although this is less pronounced than in low-risk conditions . A recen4+ T cells) that recognize H-Y minor histocompatibility antigens, which include intracellular proteins encoded by the Y chromosome of a male donor. Following H-Y antigen recognition, H-Y antibodies were observed to develop in mismatched transplantations in other organs (kidneys and hematopoietic cells) [Donor-recipient sex matching may be more significant on corneal graft outcomes in PK than in posterior lamellar keratoplasty. This could be explained in several ways. First, the thinner posterior lamellar graft has a lower antigen load; therefore, it is less immunogenic than the full-thickness graft . Second,c cells) . These ac cells) . In addiLimitations of our study include its retrospective design and the lack of HLA genetic matching data. The H-Y antigen is not present in all males, but only in those with HLA-A1. The genetic frequency of the HLA-A1 allele is population dependent and is present in 20% of the general population in the UK, 18% of the general population in Australia, and only 15% of the Jewish population in Israel . In addiIn high-risk patients, donor matching, and predominantly female-to-female donor matching, may lead to a lower corneal graft failure rate. Sex matching for every patient bares logistic difficulties; however, it may be a simple way to improve outcomes and better utilize corneal grafts in high-risk patients. We suggest that in high-risk patients where corneal graft failure is more likely, sex matching could be suggested to improve outcomes.


Leishmania, the type of host-immune response is determinant for the clinical manifestations that can lead to either self-healing or chronic disease. Multiple pathways may determine disease severity. A comparison of systemic immune profiles in patients with cutaneous leishmaniasis caused by L. guyanensis and healthy individuals with the same socio-epidemiological characteristics coming from the same endemic areas as the patients is performed to identify particular immune profile and pathways associated with the progression of disease development. Twenty-seven plasma soluble circulating factors were evaluated between the groups by univariate and multivariate analysis. The following biomarkers pairs IL-17/IL-9 , IL-17/IL-12 , IL-6/IL-1ra , IL-6/IL-12 , IL-1β/G-CSF  and IL-17/MIP-1β  showed the highest correlation mean among the patient while only INF-γ/IL-4 (ρ=0.740), 17/MIP-1β  and IL-17/IL-9  exhibited positive correlation among the control group. The cytokine IL-17 and IL1β presented the greater number of positive pair correlation among the patients. The linear combinations of biomarkers displayed IP-10, IL-2 and RANTES as the variables with the higher discriminatory activity in the patient group compared to PDGF, IL-1ra and eotaxin among the control subjects. IP-10, IL-2, IL-1β, RANTES and IL-17 seem to be predictive value of progression to the development of disease among the Lg-infected individuals.The immunopathology associated with Leishmaniasis is a consequence of inflammation. Upon infection with  Leishmania spp (L.), continues to be a public health burden in over 98 countries worldwide and still is a neglected tropical disease , severe mucosal lesions  and to life threatening visceral leishmaniasis (VL). CL and ML are also known as American Tegumentary leishmaniasis (ATL) in the American continent.Leishmaniasis, a vector-borne infectious disease caused by  disease . L.-infeL. braziliensis (Lb), L. guyanensis (Lg), L. lainsoni, L. amazonensis, L. shawi, L. naiffi and L. lindenbergi. L. braziliensis is responsible for the majority of the cases , this study attempted to find immunological pathways that could be involved in the progression to disease development in susceptible individuals and thus lead to the identification of targets for host-directed immunotherapies.To understand the immunopathogenesis of CL caused by This study was approved by the Research Ethics Committee of the Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD) and granted under the file number CAAE 09995212.0.0000.0005. All the participants or their responsible party for individuals less than 18 years old in the study provided written inform consent. Prior to the signing of the inform consent, explanation about the study was given to the participants and they were free to participate.L. guyanensis (Lg-CL) were followed at the FMT-HVD, a referral hospital for treating leishmaniasis patients. The control group comprises healthy controls (HC) living in the same endemic area as the patients, sharing similar environments.This study was conducted in the peri-rural areas of Manaus, capital of the state of Amazonas, Brazil. The patients with active CL infected with Leishmania species was performed by polymerase chain reaction (PCR) restriction fragment length polymorphism and direct nucleotide sequencing as described elsewhere , IL-13, IL-15, IL-17A, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNFα and VEGF were determined using the multiplex cytokine commercial kit Bio-PlexPro-Human Cytokine GrpI Panel 27-Plex (Bio-Rad) according to the manufacturer’s instructions in the Bio-Plex 200 Protein Array System (Luminex Corporation).Plasma levels of chemokines and cytokines from patients and control groups were compared using Kruskal-Wallis test and the graph plots were constructed using R boxblot package version 3.5.1. The interrelatedness between all biomarker was assessed using Spearman correlation test with R corrplot package version 3.5.1. Values greater or equal to 0.7 were considered positive correlation, whereas values less than or equal to -0,7 were considered negative correlation. A color map matrix and a correlation network diagram (Cytoscape version 3.8.2) were built to represent the correlations between the plasma biomarkers analyzed.Possible different biomarkers profile between patients and control groups were evaluated through Principal Component Analysis (PCA) clustering. PCA plot were generated based on the absolute abundance of biomarkers using R FactorMineR package version 3.5.1. The regions with 95% confidence levels were indicated with colored ellipses. A Linear Discriminant Analysis (LDA) were performed to determine which biomarker contributes most significantly to the discrimination of the study groups. Kruskal-Wallis (for classes) and pairwise Wilcoxon (for subclasses) tests were used to verify if the data were differentially distributed between the study groups. Logarithmic LDA score for discriminative features was built using a threshold of 2.0. The analysis was done through HutLab galaxy online platform.Heatmap was generated through hierarchical agglomerative cluster analysis to estimate the pattern of biomarker expression in the study groups. The greatest appropriate distance metric and cluster linkage were defined based on Euclidean distance and complete linkage methods, respectively. Heatmap was created using gplots package in R version 3.5.1.Lg-CL  and 376  healthy individuals with the same socio-epidemiological situations coming from the same endemicity area as the patients. The average age of the male patients with Lg-CL and male healthy controls were 39.8 ± 1.57 and 45.2 ± 1.58 years old, respectively. Age of female patient with Lg-CL and female healthy controls were34.6 ± 0.80 and 43.7 ± 1.80 years old, respectively. The skin lesions of patients were mostly located in the upper and lower limbs. All patients presented recent lesions, ranging three weeks to five weeks. The healthy individuals have no history of leishmaniasis and most of them are agriculture workers as the patients. The study population was devoid of HIV and had no history of diabetes mellitus, cardiac, renal and hepatic disease. The patients had fewer or equal to six lesions and treatment-naïve at the time of enrolment. Most of the patients had only one lesion. All the patients were first time infected. The exclusion criteria were patients with Lg-CL with previous history of leishmaniasis. Pregnant women were excluded.This study has a cross-sectional design and consisted of 354 patients with Leishmania-infected patients and healthy individuals from control group. Prior to comparison of the plasma biomarkers between patients with CL and healthy control, biomarkers were investigated if age and sex influenced the plasma levels by linear correlations using linear regressions of the R package , neutrophils (MIP-1α and IL-8), eosinophils (eotaxin), T cells and dendritic cells (RANTES). The IP-10 was the biomarker with the highest level observed in the patients. The leading role of IP-10 is the activation and maintenance of immune response by chemoattraction of monocytes, T cells, NK and dendritic cells; and the promotion of T cell adhesion to endothelial cells.The correlation analysis demonstrated a strong positive association among many biomarkers in patients with CL  seemed to be more relevant among the control subjects   –17. Recexpressed . Circulad  were observed in early lesions of patients with Lb-CL . In thisin check . CXCL10 activity . IFN-γ aactivity . Some stnfection –26. A rereatment . AdminisL. major . Immunohulocytes , 30.H1-mediated immune response , patients under remission, asymptomatic and uninfected individuals revealed molecular pathways of activation of T lymphocytes via MHC class I and type I interferon signaling besides downregulation of pathways related to myeloid cells (monocytes and neutrophils) in patients with active VL, while patients under remission showed genes correlated with activation of Notch signaling pathway and increased proportions of B cells besides T lymphocytes via MHC class I and type I interferon signaling activation as patients with active VL  . It is well-known that IL-8 attracts neutrophils at the site of infection and IFN- γ triggers macrophage activation to keep the parasites in check. Correlation of IFN- γ to IL-4 among healthy controls was also similar among HC and patients with Lg-CL (ρ = 0.7).Blood transcriptional analysis of patients with active VL caused by ctive VL . Patientignaling . Human mof IL-12 . All theectively . Anotherchagasi) . It is nLg-CL and healthy individuals sharing the same endemic area. The study is a transversal investigation and analyzed the patients only before treatment. It would be interesting to identify predictive markers of treatment failure or success by comparing the immune profile in infected individuals before and after completion of treatment.This study has few limitations. Comparisons of circulating biomarkers were performed between patients with Lg-infected susceptible individuals. While common response is observed to Leishmania-infection, however, our study points that IP-10, IL-2 and RANTES could be a predictive value of clinical parameters for progressing to disease, while PDGF, IL-1Ra and eotaxin may be protective biomarkers. IL-17 and IL-1β also are potential predictive markers for progression to disease in Lg-infected individuals. Identifying potential target in these pathways may open the way for immunotherapy.Altogether, this study identifies plasma cytokines pathways that may lead to the development of disease in The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Research Ethics Committee of the Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.TM, MS, and RR contributed for data curation. TM, JJ, MG, and RR take responsibility for the integrity of the work as a whole, from inception to published article. TM, JJ, and RR were responsible for study design and conception and drafted the manuscript. TM, JJ, LOS, GVS, FA, SP, HK, LSS, LMS, SA, KQ, JS, CS, and HS recruited healthy controls in endemic areas. TM, JJ, MG, and RR collected and cleaned the data for formal analysis. GSP and AB were responsible for statistical analysis. TM and RR interpreted the results and drafted the manuscript. All authors revised the manuscript for important intellectual content.This research was funded by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grant number 404181/2012-0 to RR, Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM), grant number 06201954/2015 to RR and FAPEAM RESOLUÇÃO N. 002/2008, 007/2018 E 005/2019 – PRÓ-ESTADO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.We thank all the patients and healthy individuals from the endemic area who have been willing to participate.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Immunohistochemical markers are associated with treatment outcome in adults with classical Hodgkin Lymphoma (cHL). Studies in children are scarce and inconsistent. We investigated in 67 children with cHL, whether the expression of CD15, CD30, PAX5, PD-1, PD-L1, CD68, CD163 and TARC at diagnosis is associated with disease free survival (DFS) and with interim remission status. Low CD15 and low TARC expression were associated with relapsed disease. Low expression of PD-L1 was associated with complete remission at interim PET-scan. Our data suggest a difference between pediatric and adult cHL. This underlines the importance of future research into specific prognostic factors in pediatric cHL, indispensable for improvement of treatment in this population. Classical Hodgkin lymphoma (cHL) contains a notably small amount of 0.1%–10% malignant Hodgkin and Reed-Sternberg (HRS) cells, surrounded by benign inflammatory cells. These cells produce different cytokines and chemokines, maintaining a specific tumor microenvironment (TME) in which the HRS cells can thrive . There iTherefore, we investigated the prognostic value of eight different immunohistochemical markers in pediatric cHL. PD-1, PD-L1, CD68 and CD163 have previously been analyzed in pediatric studies and show conflicting outcomes , 12. TARWe performed this retrospective, explorative study at the Erasmus Medical Center—Sophia Children’s Hospital (Erasmus MC—Sophia) in Rotterdam, the Netherlands.n = 23). Afterwards, fifty patients who did not experience relapse were randomly selected and included  between 2000 and 2018. Inclusion criteria were 1) children up to the age of 18 years, 2) proven diagnosis of primary cHL. Exclusion criteria were 1) nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL), 2) no representative lymphoid tissue available, 3) missing data regarding treatment outcome. An event-enriched cohort was created to increase the statistical power for our observations linked to the treatment outcome. To this end, we included all available patients with relapsed disease during the study period  using ultra view or optiview universal DAB detection Kit. In brief, following deparaffinization and heat-induced antigen retrieval the tissue samples were incubated according to their optimized time with the antibody of interest . IncubatScoring methods were based on the literature , 13, 17.The primary endpoint of this study was treatment outcome, defined as disease free survival (DFS). DFS was defined as the absence of cHL progression or relapse. Treatment failure was defined as progression or relapse of the disease at any time during the study period. First, we investigated the association between the immunohistochemical markers and DFS. Secondly, we investigated the association between immunohistochemical marker expression and early and late relapse. Early relapse was defined as relapse <1 year after finishing treatment and late relapse as relapse >1 year after finishing treatment. The secondary endpoint of this study was the achievement of complete remission at the interim PET scan. In the non-European Network-Paediatric Hodgkin Lymphoma-C1/C2 (EuroNet-PHL-C1/C2) treatment protocols, instead of a PET scan, ultrasound evaluation or a computed tomography (CT) scan was used for interim evaluation , 21. A sp-value of <0.05 in the univariate analysis was considered statistically significant. IBM SPSS Statistics for Windows, Version 25.0  and R software  were used for the statistical analysis.In order to study the univariate prognostic value of potential predictors, we applied the rule of thumb to reassure that we included at least 10 cases per variable. To examine the differences in patients’ characteristics at baseline the Pearson Chi-Square test, the Fisher’s exact test, the Independent Student’s t-test, and the Mann-Whitney test were used. For the primary outcome analysis, we performed the Mann-Whitney to investigate if there were differences in expression patterns between the different outcome groups. Afterwards, we performed receiver operating characteristics (ROC) analysis to check the discriminating power for DFS of each biomarker. For the secondary outcome analysis, we also investigated if there was an association between the different markers by Mann-Whitney test and we performed ROC curves to investigate the discriminative power for early versus late relapse or remission status. For remission status, a sub-analysis was performed for patients treated according to the EuroNet-PHL-C1/C2 treatment protocols. A 2-tailed Seventy-three patients were included in the study. Six patients were excluded, based on the exclusion criteria . The meap = 0.012). Patients with lower stage were more likely to achieve CR at interim PET-scan (p = 0.033).Thirty patients (44.8%) did not achieve complete remission (CR) at interim PET scan. Significantly more females than males achieved CR  (The Mann-Whitney test showed that the percentage of CD15 and TARC expression on HRS cells were associated with DFS. Patients with treatment failure had a significantly lower percentage of CD15 expression on HRS cells (p 0.002) and a significantly lower percentage of TARC expression on HRS cells (= 0.019) . For the= 0.019) . After t= 0.019) . The per= 0.019) . PD-L1 eThere was not enough power to perform a sub-analysis with only the patients treated according to the EuroNet-PHL-C1/C2 treatment protocols. The sub-analysis to investigate the association between the immunohistochemical markers and early versus late relapse showed no association or discriminative power for any of the markers .n = 45), showed that expression of PD-L1 in the TME was associated with remission status. The Mann-Whitney test showed that patients with complete remission on interim PET-scan had significantly lower PD-L1 expression on the TME than patients with no complete remission at interim PET (p = 0.04)  . The ROC = 0.04) . The othThe results of our study add significantly to the limited data available for pediatric cHL patients –10, 12. We found that low CD15 expression on HRS cells at diagnosis was associated with relapsed disease. Previous studies described that loss of CD15 expression at diagnosis was associated with overall survival (OS) and failure-free survival (FFS) in pediatric patients and in adult patients , 24, 25.We found that low expression of TARC on HRS cells was associated with treatment failure, although the discriminative power of TARC was limited. The impact of TARC expression on HRS cells has not been described in the literature yet. However, it is known that TARC plays a crucial role in the pathogenesis of cHL . The sec+ T cells and CD8+ T cells which augments the immunosuppression  binding to CD30) , 37. RecOur study is the first to examine eight different markers simultaneously. Another strength of our data is the event-enriched setting particularly in pediatric cHL patients. However, due to the low relapse rate in cHL, we included patients diagnosed over a period of 18 years and therefore treated with different treatment regimens. Despite this event-enriched cohort, the study still lacked the statistical power to perform a sub-analysis per treatment protocol for the primary outcome. Another possible limitation of the study is the use of immunohistochemical staining. This can lead to inconsistent results depending on differences in tissue fixation, the type of antibody and staining method, the scoring method, and the observer’s interpretation. To minimize these inconsistencies, we based our scoring method on published previous studies , 13, 17 In conclusion, we found that low CD15 and TARC expression on HRS cells are associated with treatment failure. Furthermore, we found that PD-L1 expression in the TME at diagnosis is associated with CR status at interim analysis. Both these outcomes should be further investigated. Furthermore, our data demonstrate differences in the expression patterns and prognostic impact of immunohistochemical markers between pediatric and adult patients with cHL. Further research into these differences may lead to specific prognostic factors in pediatric cHL, indispensable for improvement of treatment in this population.


Cervical cancer screening participation is suboptimal in most settings. We assessed whether human papillomavirus (HPV) self-sampling may increase screening participation among long-term non-attenders in Norway.A pragmatic randomised controlled trial with participation as the primary outcome was initiated in the national cervical screening programme in March 2019. A random sample of 6000 women aged 35–69 years who had not attended screening for at least 10 years were randomised 1:1:1 to receive either (i) a reminder to attend regular screening (control), (ii) an offer to order a self-sampling kit (opt-in) for HPV testing or (iii) a self-sampling kit unsolicited  for HPV testing.P < 0.0001; participation difference (%) send-to-all vs. control: 22.9 ; opt-in vs. control: 12.3 ; send-to-all vs. opt-in: 10.7 ). High-risk HPV was detected in 11.5% of self-samples and 9.2% of clinician-collected samples (P = 0.40). Most women (92.5%) who returned a positive self-sample attended the clinic for triage testing. Of the 933 women screened, 33 (3.5%) had CIN2 + , and 11 (1.2%) had cervical cancer .Total participation was 4.8%, 17.0% and 27.7% among control, opt-in and send-to-all (Opt-in and send-to-all self-sampling increased screening participation among long-term, higher-risk non-attenders.ClinicalTrials.gov NCT03873376. Screening has reduced cervical cancer incidence  and mortApproximately 17% of women aged 35–69 years in Norway have not been screened for at least 10 years . Non-attHPV infection is a necessary cause of cervical cancer . PersistStudies on the effect of HPV self-sampling on participation among under-screened women often include women only slightly overdue for screening, who may be easier to engage than long-term non-attenders, while few randomised controlled trials have by design specifically targeted long-term non-attenders , 19–22. The primary aim of this study was to compare cervical cancer screening participation among women in Norway who have not attended screening for at least 10 years, who received either a standard reminder letter to attend screening in a clinic (control arm), an offer to order an HPV self-sampling kit (opt-in arm), or were sent an unsolicited HPV self-sampling kit . As secondary endpoints, we estimated corresponding hrHPV positivity rates and occurrence of CIN2+ among the long-term non-attenders who participated in screening, and attendance to follow-up.The Cancer Registry of Norway (CRN) is responsible for the national cervical cancer screening programme , and invites women aged 25–69 years to attend screening. The programme is currently transitioning from cytology every 3 years to HPV primary screening every 5 years for women aged 34–69 years. CervicalScreen Norway uses a centralised invitation procedure and issues standard open reminder letters to women who have not been registered with a screening test during the recommended interval. A second reminder is issued after one year if a woman is still not registered with a test result. These reminders encourage women to schedule an appointment with their GP or a gynaecologist for a screening test. All cytology and HPV screening results, as well as any associated histology results, are registered in the CervicalScreen Norway database. All records are associated with the personal identity number unique to each Norwegian resident.The target population was women residing in the counties of Hordaland, Rogaland, Sør-Trøndelag and Vest-Agder who had not participated in the screening programme for the last 10 years. These regions consist of mixed rural and urban areas and cover about one-third of the Norwegian population. During the study, primary screening was offered by HPV test in Hordaland, Rogaland and Sør-Trøndelag, and by cytology in Vest-Agder. A priori power calculations showed that we needed at least 1417 women in each intervention arm to detect a difference of 5 percentage points between the control arm and the intervention arms with 90% power. Using the CervicalScreen Norway database, we identified 28,125 women who were eligible for the study, of which 6000 (21.3%) were randomly selected for invitation to the study. The randomly selected women were individually randomised 1:1:1 into the control, opt-in or send-to-all intervention arm without further restrictions. The eligible women were unaware of the randomisation. Blinding was not possible due to the nature of the interventions. Randomisation procedures were performed using the sample function in Stata by a programmer who was not involved in the conduct of the trial. Invitations were sent during March–August 2019.P = 0.4), were excluded from the study due to: (i) incorrect address, (ii) active refusal to participate in the study, or (iii) ineligibility for the study (living abroad/had prior hysterectomy/had a screening test between study sampling and invitation)  invited women, similarly distributed by intervention arm; 108 (5.4%) in the control arm, 103 (5.1%) in opt-in arm, and 122 (6.1%) in the send-to-all arm; Women in the control arm received a CervicalScreen Norway reminder letter to attend the regular screening . The letter also stated that they were randomly selected to participate in a study aiming to improve cervical cancer prevention. The letter included no information about self-sampling.Women in the opt-in arm received an invitation letter to order a self-sampling kit. The women could order by mail, e-mail, or through a webpage. The invitation included a unique order code, information about the study, a reply slip, and a prepaid envelope to return the order. A dedicated webpage for ordering a self-sampling kit was available in Norwegian, English, French, Spanish, Polish, Turkish and Arabic. Each order code could only generate one web order.A self-sampling kit was mailed unsolicited to women in the send-to-all arm, together with the study invitation letter. The content in the opt-in and send-to-all invitation letters was the same (apart from the description of the ordering procedure in the opt-in letter) and included information that the women alternatively could schedule a regular screening appointment with their GP if they did not want to use the self-sample.Similar to regular CervicalScreen Norway reminders, all invitation letters contained general screening information. The letters were on CervicalScreen Norway stationary and were signed by the CervicalScreen Norway head. In addition, letters contained study-specific information, a link to the study webpage, and information that they could refrain from participating in the study. All invitation letters were in Norwegian, except for an English reference to a webpage. The study webpage was available in Norwegian and English and included a link to an instructional video on how to collect a self-sample with options for Norwegian or English audio, and English, French, Spanish, Polish, Turkish or Arabic subtitles.The kit included the self-sampling device with user instructions, a sealable plastic bag, a prepaid return envelope addressed to the laboratory, and an information leaflet. The Evalyn® Brush (dry brush)  was used for self-sampling in this study. The self-sampling device had a hidden radio frequency identification chip containing a pseudonymized number unique to each invited woman and had no visible personal information. Women returned the self-sample in the plastic bag and mailed it in the return envelope. Women who had not ordered/returned a self-sample within 3 weeks received one reminder letter to order/return the self-sample. The self-sampling kit was free of charge for the women.All self-samples were analysed at the Norwegian HPV reference laboratory at Akershus University Hospital. Soon after receipt at the laboratory, the brush tip was removed from the self-sampling device, transferred to a vial with 4.5 mL ThinPrep PreservCyt and stored at room temperature for at least 24 h. During storage, tubes with the brush tip were vortexed three times for 15 s to ensure proper dissolution of the sample material. The samples were tested for hrHPV using the Cobas® 4800 HPV test , which individually reports HPV16 and HPV18 results, in addition to a pooled result of the 12 hrHPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. Beta-globin is included as internal quality control in a fourth channel. Samples with an invalid test result at the first run were retested, which gave a valid test result in every case. Analysis was completed within 21 days after receipt for all samples.N = 218, 91.6%) or cytology .Clinician-taken screening samples were processed according to CervicalScreen Norway guidelines . They weAll women who returned a self-sample were informed about their test result per ordinary mail within 6 weeks after receipt of the self-sample in the lab. Women with an HPV-negative result were encouraged to continue attending the regular cervical cancer screening programme at the recommended interval. Women with a hrHPV-positive self-sample received the result together with a pre-booked triage appointment with a physician. To address the feasibility and performance of alternative triage strategies that could be implemented in a screening programme, women were sequentially allocated to triage at their GP or a gynaecologist practising in the largest city in their county. The notification letter to women in each group was identical. Physicians were informed that the patient had tested positive for HPV after participating in a study offering HPV self-sampling to long-term non-attenders. Women referred to GP triage underwent cytology, while women referred to gynaecologist triage underwent further gynaecological examination, which always included cytology and colposcopy, and biopsy if deemed necessary. To simulate a routine healthcare setting, the women paid the deductible for the GP or the gynaecologist appointment themselves , as they would in the CervicalScreen Norway. Management after the scheduled triage visit of women who had a positive hrHPV self-sample was not specific for the study and thus follow national guidelines . SimilarData regarding clinician-collected screening tests (including triage tests taken outside the study) was taken from the CervicalScreen Norway database. Data on histological outcomes from colposcopy referrals were taken from the CervicalScreen Norway and the CRN databases. Dates were delivered by month and year. If several histological diagnoses were available for the same women, we only considered the most severe diagnosis. The last registry linkage was performed in December 2020, which allowed at least 16 months of registry follow-up after the study invitation. Histology diagnoses were interpreted according to WHO guidelines . Income P values refer to chi-squared tests, or the Fisher´s exact test for observed counts <5. For comparisons of central tendency, P values refer to the Mann–Whitney U test. Homogeneity of variance was assessed by Levene’s test. All tests were two-sided and P values < 0.05 were considered statistically significant. Analyses were performed using Stata version 17MP or R version 3.5.3.Screening participation was defined as returning a valid self-sample or having a clinician-collected screening test within 6 months after receipt of the invitation letter. Attendance to triage was assessed among women with a hrHPV-positive self-sample and was defined as attending at the allocated physician  or an unallocated physician within 6 months of notification of a positive self-sample. Women who returned a hrHPV-positive self-sample and who were not registered with a cytology test by 3 months following the scheduled appointment received a reminder from the CervicalScreen Norway encouraging them to order an appointment at their GP. Screening participation among women in the control arm was only possible by attendance to a clinician. For each self-sampling arm, we present data separately and in total for women who self-sampled and women who had a clinician-collected screening test. The main analyses of participation, which is the trial´s primary outcome measure, are intention-to-treat . We also provide per protocol analyses for the participation differences as a supplement. We present numbers and proportions of screening participation by intervention arm  overall, by age groups  and by screening history . Differences in screening participation among intervention arms are presented as absolute participation differences  and as relative participation differences  with associated 95% confidence intervals (95% CI). Further, we present numbers and proportions of hrHPV-positive screening tests by intervention arm. We present attendance to triage by self-sampling arm and by mode of triage  for women with a hrHPV-positive self-sample. Since positive clinician-collected screening tests do not require a separate triage visit, we present attendance to the CervicalScreen Norway recommended follow-up after a positive reflex triage test for women in the control arm and women in the self-sampling arms who screened clinically. Numbers and proportions of histologically verified high-grade lesions are presented by intervention arm. To investigate the diagnostic yield by mode of triage, we also present the histologically verified high-grade lesions separately for women triaged by study gynaecologists, women triaged by study GPs, and women who had a clinician-collected screening test or attended triage outside the study . For comparisons of counts and proportions, P = 0.94), time since last screen (P = 0.99), county of residence (P = 0.96) and income (P = 0.28) (Table All baseline characteristics addressed in the study cohort were similarly distributed by the study arm Table . The mea8) Table .Table 1BP < 0.0001, Fig. P = 0.33, Table Total participation  was 4.8% in the control arm, 17.0% in opt-in arm and 27.7% in send-to-all arm  women ordered a self-sample, of which 144 (35.7%) ordered after receiving a reminder, and 250 (13.2%) returned the self-sample for analysis. Opt-in orders were made by ordinary mail (51.6%), web (33.0%), e-mail (14.2%) or telephone to the study centre . Among 445 women in the send-to-all arm who submitted a self-sample, 254 (57.1%) did so after receiving a reminder.P < 0.0001). Also, the RPD was highest among never-screeners, at 6.1 (95% CI 3.4–11.1), 13.4 (95% CI 7.5–23.8) and 2.2 (95% CI 1.7–2.8) for opt-in vs. controls, send-to-all vs. controls and send-to-all vs. opt-in, respectively . Among controls, 6.0% were positive for any hrHPV, which was non-significantly lower than observed among women in the opt-in (10.8%) and send-to-all (11.8%) intervention arms (P = 0.28)  Table .Table 3PP = 1), respectively, for scheduled attendance, and 92.6% and 92.5% (P = 1), respectively, for any attendance within 6 months , respectively. However, among women who were biopsied and had a histology result, the median (IQR) period between the positive self-sample and the histological diagnosis was longer for women who were allocated to and attended GP triage than for women who were allocated to and attended gynaecologist triage, at 4  and 3  months, respectively (P = 0.01). Among women who screened clinically , and who had a positive reflex triage result leading to direct referral to colposcopy with biopsy or a new HPV test after 12 months , and in 1.1%, 3.7% and 3.8% of the screened women (P = 0.52) in the control, opt-in, and send-to-all arms, respectively. A total of 0, 4 and 7 cases of cervical cancer were detected in the control, opt-in and send-to-all arms, respectively, giving corresponding detection rates per woman screened of 0%, 1.2% and 1.3% (P = 0.81). In each of the self-sampling arms, the detection rate of CIN2+ and cancer per woman screened was similar among women who screened by self-sampling and women who attended screening at a clinic (Table A total of 1, 12 and 20 prevalent cases of CIN2+ were detected in the control, opt-in, and send-to-all arms, respectively Table . Thus, wen P = 0.2 in the A relatively high proportion of women with screening-positive tests were subsequently diagnosed with CIN2+ Table . AlthougWe show that offering HPV self-sampling to long-term non-attending women as opt-in or as send-to-all significantly increases cervical screening participation relative to the standard reminder letter, and that send-to-all gives the largest increase in participation.Although the effect of self-sampling interventions may differ between studies due to differences in study design and screening setting, increased participation by the send-to-all self-sampling strategy is well established compared to clinician-collected screening , 26. In Characteristics of the targeted screening population are important for the way in which self-sampling interventions may affect participation. When self-sampling is offered more widely to women who are overdue for screening, it tends to replace screening at a clinic in addition to increase overall participation , 22. We Although inferior to send-to-all, we found that opt-in also increased screening participation when compared to controls who received the routine reminder letter. Thus, this type of intervention should not be dismissed, although opt-in in other settings often did not improve participation beyond a routine reminder letter to screen . The wayLong-term non-attending women may be hesitant to undergo a gynaecological examination for screening purposes , but ourThe hrHPV positivity rate of the total study population of long-term non-attenders was nearly twice as high as the corresponding rate among women of the same age in the ordinary screening population . In bothDue to low numbers, the CIN2+ occurrence observed here should be interpreted with caution, especially in terms of comparisons between intervention arms or other subgroups. Among all the long-term non-attending women who were screened during the study, 3.5% and 1.2% were diagnosed with CIN2+ and cervical cancer, respectively, while the corresponding rate among women aged 34–69 years in the CervicalScreen Norway is 1.2% and 0.1% [GP and gynaecologist triage of long-term non-attending women who had a hrHPV-positive self-sample gave similar attendance and diagnostic yield. However, for long-term non-attending women who have CIN2+ , it is important to be treated quickly, and our results clearly showed that the final diagnostics appeared earlier if women were referred directly to a gynaecologist who could perform a colposcopic biopsy at the triage visit than if they were referred to their GP for cytology triage. Furthermore, the CIN2+ and CIN3+ detection rates among long-term non-attenders with a positive screening test exceeded the risk thresholds for which colposcopy usually is considered good practice , 32. SimA strength of this study is the use of national registry data that ensures a precise definition of the study cohort and complete information on participation and diagnostics. Targeted sampling of long-term non-attenders also ensures a sufficient sample size for robust inference regarding comparisons of participation by the study arm in this part of the screening population. Another strength is the randomised design that enhances the representativeness of the study sample and the similarity of the intervention arms. The intervention arms should thus be highly comparable, and the results of the present study should have high generalisability to the population of long-term non-attenders in Norway. Furthermore, we used a self-sampling device and a PCR-based HPV DNA test that were clinically validated and gave no invalid self-samples in this study. Finally, the trial was embedded in the CervicalScreen Norway, which should make the study results relevant to a real-world scenario where self-sampling is offered as part of an organised programme. However, the invited women were informed that this was a study, which could have influenced their motivation to participate. A further limitation is the relatively low subgroup numbers regarding hrHPV positivity, attendance to triage and CIN2+ occurrence, which limits the precision of the inference that can be made regarding these secondary outcomes of the study.We conclude that opt-in and send-to-all self-sampling strategies increase screening participation among long-term non-attenders to cervical screening in Norway. If implemented as part of the screening programme, both strategies would probably improve secondary prevention of cervical cancer and thus benefit women’s health. Long-term non-attenders also have an elevated risk for hrHPV infection and high-grade cervical lesions, which highlights the large potential to improve cervical cancer prevention by increasing the screening participation of this population. The send-to-all strategy would maximise the preventive effect because it clearly increased participation the most. Maintaining an offer for clinician-collected screening will still be important, since some long-term non-attenders offered self-sampling preferred this option. We also show that management of hrHPV-positive self-samples by GP cytology triage or direct referral to colposcopy by gynaecology specialists give similar results in terms of triage attendance and diagnostic yield. Our study supports findings that direct referral to colposcopy might be the best option for hrHPV-positive women in this population, especially if they are positive for HPV16/18, because they are at a relatively high risk for sequelae and colposcopy referral gives the shortest lag to a histologically confirmed diagnosis and treatment. Colposcopy is generally a safe procedure, thus over-referral will not be a burden for the patient.The opinions expressed by the authors are their own, and this material should not be interpreted as representing the official viewpoint of the U.S. Department of Health and Human Services, the National Institutes of Health, or the National Cancer Institute.Supplementary figure legendsSupplementary figurePer protocol analysis


During the trial production of Armillarisin A for injection (AA-I), unidentified needle-like yellow-brown crystals were occasionally observed. Here, we report an ultra-high performance liquid chromatography–tandem mass spectrometry (UPLC-MS) method for determining the source of the visible foreign bodies in the formulations of Armillarisin A active pharmaceutical ingredient (AA-API). AA-API, photolyzed samples, the intermediate polymer, and the excipient analyzed determined after the separation on a Waters Symmetry C18  column with a mobile phase consisting of a methanol/acetic acid (0.1 mol/L) aqueous solution (50:50). Furthermore, the crystal type of the visible foreign bodies, the intermediate polymer and AA-API were investigated by X-ray powder diffraction (XRD). The results revealed that the characteristics of the visible foreign solids were the same as those of AA-API as regards UPLC peak position (368 nm) and MS spectrum in negative ion detection mode. The visible foreign solids were thus identified as unpolymerized crystals of AA-API and were attributed to AA-API itself. The results showed that the production process could be improved by changing the stirring method and frequency as well as by optimizing the polymerization temperature to ensure the safety, stability, and control of the product quality in the stage of batch production. Most commercially available AA-I contain pharmaceutical-grade propylene glycol as a solvent. When used in high doses, it sometimes causes propylene glycol toxicity [-2002] ofulations . There iThe UPLC chromatograms (368 nm) of AA-API, a photolyzed sample, visible foreign solids, intermediate polymer, and blank excipient are shown in - at m/z 233 were further performed, and the ESI-MS2 scan mass spectra . The solvent used in the preparation of standard AA-I can vary. However, the detailed formulation and process parameters are commercial secrets belonging to the producing enterprises. They are generally not shared with the public but only with the national regulatory authorities.According to the formulation composition and production process recorded in the Chinese national pharmaceutical standard [No. WS-10001-(HD-1207)-2002] of AA-I, a trial production was carried out. Pharmaceutical-grade povidone K30 was chosen as a cosolvent, as it provided more advantages than those obtained with different cosolvents by other manufacturers, such as high solubility and stability. The samples used in this study were all from the trial production batch ; those containing visible foreign bodies were collected for further investigation.Based on the trial production process, AA-API, the polymerization agent (povidone K30), the solvent , the surfactant (polysorbate 80), and the antioxidant (thiourea) were employed. Among these compounds, only Armillarisin A and povidone K30 are crystalline. Owing to the formulation composition, the production process, and the type and structural characteristics of the raw and auxiliary materials used in the Chinese national pharmaceutical standard [No. WS-10001-(HD-1207)-2002] of AA-I, the visible foreign solids may derive from either API (Armillarisin A) or the polymerization agent (povidone K30), but not from the solvent , the surfactant (polysorbate 80), or the antioxidant (thiourea).Furthermore, AA-I can be decomposed by various factors such as light, high temperature, and pH ,11. The + in negative ion detection mode, thus forming the quasi-molecular ion [M-H]- at m/z 233.08 . M1 was cleaved in turn, losing a molecule of CH2=C=O, and CO yielded the fragment ions at m/z 161.01 and m/z 135.01. The quasi-molecular ion [M-H]- at m/z 233 lost a molecule of CO2, yielding a fragment ion t m/z 189.02 (M2). M2 could lose a molecule of H2O, yielding a fragment ion with an m/z of 171.02, a molecule of CH2=C=O, yielding a fragment ion at m/z 147.01, or a molecule of HCHO, yielding a fragment ion at m/z 158.95. The quasi-molecular ion [M-H]- at m/z 233 lost one molecule of H2O to form a fragment ion at m/z 214.99 (M3); the quasi-molecular ion [M-H]- at m/z 233 lost one molecule of CH2=C=O to form a fragment ion at m/z 191.02 (M4). M4 lost one molecule of H2O, producing a fragment ion at m/z 162.99, and the further loss of one molecule of H2O produced a fragment ion at m/z 147.01.The peak position of the visible foreign solids was consistent with that of AA-API, according to the respective UPLC chromatograms and MS spectra . The visXRD analysis was performed in the 3–90° range . The resThe crystal type of the visible foreign bodies was very different from that of the intermediate polymer and of AA-API . The posMethanol (HPLC grade) was purchased from Scharlau , acetic acid (HPLC grade) was purchased from Aladdin Holdings Group Co.  and ammonia was obtained from Zhejiang Hanno Chemical Technology Co. Ltd. . Purified water was prepared by a Milli-Q water purification system . The reagents and excipients used in AA-I production, such as povidone K30, were of medicinal grade and met the requirements of the Chinese Pharmacopoeia or the national drug quality standards.The photolytic samples were prepared by spreading AA-API  in a dish and then placing it in a lightbox (6000 Lux) for 24 h; it was finally stored in a closed and shaded environment. The intermediate polymer was prepared by mixing AA-API and povidone K30 in appropriate amounts, and the intermediate polymerization was performed according to the Chinese national drug standard of AA-I. AA-API (12.5 mg), the photolyzed samples (12.5 mg), and the intermediate polymer (12.5 mg) were separately placed in volumetric flasks (50 mL), with methanol added to adjust the volume. A sample with a concentration of 5 μg/mL was obtained by pipetting 1 mL of the solution into a 50 mL volumetric flask and adjusting the volume with methanol.The blank excipient was prepared from anhydrous ethanol, N, N-dimethylacetamide, propylene glycol, polyethylene glycol 400, polyethylene glycol 6000, polysorbate 80, and thiourea, according to the preparation method of AA-I recorded in the Chinese national drug standards . The blaAs reported in the Chinese national drug standard of Armillarisin A injection, an ammonia solution was used for a color reaction. Thus, the ammonia solution was used to try to dissolve the visible foreign bodies which were insoluble in water. The ammonia test solution was prepared by adding concentrated ammonia (400 mL) to a volumetric flask (1000 mL), and the volume was adjusted by adding water. Due to the stability test results that showed that two degraded components could be observed 2 h later under the same UPLC conditions, the ammonia solution was prepared only before use in this study as a cosolvent.Some AA-I formulations were selected from the trial production and left standing for 2 min. A syringe was then used to aspirate the upper clear layer, while the large needle-like crystals at the bottom were retained. The blank auxiliary solution (0.5 mL) was aspirated by another syringe to rinse the inner wall of the ampoule, the ampoule was shaken slowly and left standing still until the upper layer solution was clear and could be aspirated. This process was repeated twice, and a change in the crystal properties during this process was observed. Methanol (0.5 mL) was then aspirated to rinse the inner wall and the needle crystals in the same way. This process was repeated twice, and the solution was left standing while the upper clear layer was aspirated. An ammonia/water  solution was added to the residual crystals, the mixture was shaken until the crystals disappeared, and then it was diluted with methanol to obtain the unidentified visible crystal sample solution (1 mL).The samples were accurately weighed using a QUINTIX35-1CN 1-in-100,000 electronic balance .Ultra-high performance liquid chromatography analysis was performed on an Acquity I-Class PLUS ultra-high performance liquid chromatographer (Waters) with an Acquity/Xevo TQS ultra-high performance liquid chromatographer–mass spectrometer (Waters) coupled with an electrospray ionization source, in negative ion detection mode. The source temperature was 150 °C, the capillary voltage was 2 kV, the cone voltage was 30 V, and the cone hole gas flow rate was 150 L/hr. The shell gas temperature was 350 °C and the shell gas flow rate was 650 L/hr. The carrier gas flowed at 0.7 MPa at a collision energy of 25 V. The separation was performed on a Waters Symmetry C18 column  with a methanol −0.1 mol/L acetic acid solution (50:50) at a flow rate of 0.4 mL/min and a column temperature of 25 °C.The XRD analysis was performed on a RIKEN X-ray multi-crystal diffractometer (XRD Rigaku Smart-Lab 9 kW) at an operating capacity of 1.6 kW  within the scanning range of 3~90° at a step distance of 0.02. The target material was Cu.In conclusion, this study enabled the qualitative identification of the visible foreign bodies, occasional needle crystal-like yellow-brown precipitates, observed in the trial production of AA-I samples, by comparing the UPLC-MS chromatograms and spectra of the visible foreign solids with those of AA-API, photolyzed samples, the intermediate polymer, and the excipient, as well as by combining the LC-MS/MS and XRD information. The results revealed that the visible foreign solids consisted of a small amount of AA-API ingredients not encapsulated by the povidone K30 copolymer. These results can be beneficial for improving the production technology and the quality control in drug production enterprises and can help quickly solve similar problems that may occur in production processes. To sum up, optimizing the preparation temperature, changing the stirring method and the stirring frequency, and conducting a verification process can further improve a product quality and ensure its safety for clinical use .There is a patent named “A method for identifying endogenous visible foreign bodies in Armillarisin A injection”, with registration No. 202211654876.9, resulting from the work reported in this manuscript.